Immunoglobulins

Title: Immunoglobulins.Abstract: The present invention discloses humanised anti-IL-18 antibodies, methods of manufacture, and methods of treatment with said antibodies. Further disclosed are screening methods using for example surface plasmon resonance to identify antibodies with therapeutic potential. ...

This application is a continuation of U.S. patent application Ser. No. 11/752,707 filed May 23, 2007 which in turn, claims foreign priority to Great Britain Application Number 0610438.4 filed May 25, 2006 and Great Britain Application Number 0611046.4 filed Jun. 5, 2006.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunoglobulins such as antibodies and in particular to humanised antibodies, useful in the treatment and diagnosis of conditions mediated by human interleukin-18.

TH1 cells, which produce proinflammatory cytokines such as IFN-γ, IL-2 and TNF-β (Mosmann, et al., J. Immunol. 136:2348, 1986), have been implicated in mediating many autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (RA), type 1, or insulin dependent, diabetes (IDDM), inflammatory bowel disease (IBD), and psoriasis (Mosmann and Sad, Immunol. Today 17:138, 1996). Thus, antagonism of a TH1-promoting cytokine such as IL-18 would be expected to inhibit disease development. Il-18 specific mAbs could be used as an antagonist.

The role of IL-18 in the development of autoimmune diseases has been demonstrated. Accordingly, it has been demonstrated that IL-18 expression is significantly increased in the pancreas and spleen of the nonobese diabetic (NOD) mouse immediately prior to the onset of disease (Rothe, et al., J. Clin. Invest. 99:469, 1997). Similarly, IL-18 levels have been shown to be markedly elevated in the synovial fluid of rheumatoid arthritis patients (Kawashima, et al., Arthritis and Rheumatism 39:598, 1996). Furthermore, it has been demonstrated that IL-18 administration increases the clinical severity of murine experimental allergic encephalomyelitis (EAE), a Th1-mediated autoimmune disease that is a model for multiple sclerosis. In addition, it has been shown that neutralizing anti-rat IL-18 antiserum prevents the development of EAE in female Lewis rats (Wildbaum, et al., J. Immunol. 161:6368, 1998). Accordingly, IL-18 is a desirable target for the development of a novel therapeutic for autoimmunity.

Taniguchi, et al., J. Immunol. Methods 206:107, describe seven murine and six rat anti-human IL-18 monoclonal antibodies (mAbs), which bind to four distinct antigenic sites. One of the murine mAbs (#125-2H), and the six rat mAbs inhibit IL-18-induced IFN-γ production by KG-1 cells, with the rat mAbs exhibiting neutralizing activities 10-fold lower than that of #125-2H. As demonstrated by Western blot analysis, three of the murine mAbs, but none of the rat mAbs, are strongly reactive with membrane-bound human IL-18. In addition, an enzyme-linked immunosorbent assay (ELISA) to detect human IL-18 is described, utilizing #125-2H and a rat mAb. The limit of detection of this ELISA is 10 pg/ml.

European patent application EP 0 712 931 discloses two mouse anti-human IL-18 mAbs, H1 (IgG1) and H2 (IgM). As demonstrated by Western blot analysis, both mAbs react with membrane-bound human IL-18, but not with membrane-bound human IL-12. HI is utilized in an immunoaffinity chromatography protocol to purify human IL-18, and in an ELISA to measure human IL-18. H2 is utilized in a radioimmunoassay to measure human IL-18.

Neutralizing IL-18 antibodies may potentially be useful in relieving autoimmune diseases and related symptoms in man. Hence there is a need in the art for a high affinity IL-18 antagonist, such as a neutralizing monoclonal antibody to human interleukin 18, which would reduce Th1 cell differentiation and proliferation and thus autoimmune diseases and related symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of temperature on the on-rate (ka) of H1L1 and H1L2.

FIG. 2 shows the effect of temperature on the off-rate (kd).

FIG. 3 shows the effect of temperature on the equilibrium constant (KD).

FIGS. 4A-4C show representative data from one experiment that generated the EC50 values illustrated in Table 7.

FIGS. 18A and 18B show the results of binding ELISAs using H1L2 and C10, respectively, to human IL-18-bound IL-18BP.

SUMMARY

OF THE INVENTION

In one aspect, the present invention provides a humanised anti-interleukin-18 antibody comprising a heavy chain and light chain having the following complementarity determining regions (CDRs):

CDRH1: SEQ ID NO:1;

CDRH2: SEQ ID NO:2;

CDRH3: SEQ ID NO:3;

CDRL1: SEQ ID NO:4;

CDRL2: SEQ ID NO:5; and

CDRL3: SEQ ID NO:6.

In a second aspect, the present invention provides a humanised anti-interleukin-18 antibody comprising a heavy chain and light chain having the following CDRs:

CDRH1: SEQ ID NO:1;

CDRH2: SEQ ID NO:2;

CDRH3: SEQ ID NO:3;

CDRL1: SEQ ID NO:4;

CDRL2: SEQ ID NO:5; and

CDRL3: SEQ ID NO:6

wherein the residue at position 71 of the light chain is substituted by the corresponding residue found in the donor antibody from which the CDRs are derived.

It will be apparent to those skilled in the art that the term “derived” is intended to define not only the source in the sense of it being the physical origin for the material, but also the material that is structurally identical to the material, but which does not originate from the reference source. Thus, the corresponding residue “found in the donor antibody framework from which the CDRs are derived” need not necessarily be purified from the donor antibody framework. Similarly, CDRs “derived from a donor antibody” need not necessarily be purified from the donor antibody.
CDRs and framework regions (FR) and numbering of amino acids follow, unless otherwise indicated, the Kabat definition as set forth in Kabat, et al., “Sequences of immunological interest”, NIH.

In a third aspect, this invention provides a humanised anti-interleukin-18 antibody comprising CDRs derived from a donor antibody grafted onto a human acceptor framework which anti-interleukin 18 antibody comprises CDRs having the sequences set forth in SEQ ID 1, 2, 3, 4, 5, and 6, wherein the residue at position 71 of the light chain of said anti-interleukin-18 antibody is identical to the residue found in the corresponding position in the donor antibody framework.

In a fourth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising CDRs having the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, wherein the antibody comprises a tyrosine at position 71 of the light chain.

In a fifth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising a heavy chain having CDRs set forth in SEQ ID NOs: 1, 2, and 3, and a light chain having CDRs set forth in SEQ ID NOs: 4, 5, and 6, wherein said light chain CDRs are derived from a donor antibody having a tyrosine at position 71 of the donor antibody light chain.

In a sixth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising CDRs from a donor antibody and a tyrosine at position 71 of the light chain of said humanised antibody, wherein the donor antibody is 2C10 or a framework variant thereof (i.e., the humanised antibody comprises the same CDRs but a different framework as 2C10. See U.S. Pat. No. 6,706,487).

In a seventh aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

(a) a heavy chain having CDRs with the sequences set forth in SEQ ID NOs:1, 2, and 3 grafted onto a human heavy chain acceptor framework; and

In an eighth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

(a) a heavy chain having CDRs permissive of specific binding to human IL-18; and

(b) a light chain comprising an acceptor framework and having CDRs with the sequences set forth in SEQ ID NOs: 4, 5, and 6 and having a tyrosine residue at position 71.

In one embodiment of the invention, the CDRs of the light chain are located at positions within the acceptor framework that correspond to the respective positions of the sequences set forth in SEQ ID NOs: 4, 5, and 6 within the sequence set forth in SEQ ID NO:35. In another embodiment of the invention, the light chain and/or the heavy chain are non-immunogenic in a human patient.

In a ninth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

(b) a human acceptor framework, wherein said acceptor framework comprises at position 71 of the light chain acceptor framework a different type of aromatic amino acid from the aromatic amino acid in part (a); and

(c) wherein the anti-interleukin-18 antibody comprises a light chain having at position 71 an aromatic amino acid derived from the antibody of part (a).

In a twelfth aspect, this invention provides a humanised anti-interleukin-18 antibody, wherein said antibody displays a equilibrium constant (KD) of 300 pM or less with respect to binding of human IL-18 when measured by surface plasmon resonance (e.g., Biacore™, using a Biacore™ 3000 instrument and conditions as set out in Example 4.a. below) at 37° C.).

In a thirteenth aspect, this invention provides a humanised anti-interleukin-18 antibody, wherein said antibody comprises CDRs as set forth in SEQ ID NO:1, 2, 3, 4, 5, and 6 and displays a equilibrium constant (KD) of 300 pM or less with respect to binding of human IL-18 when measured by surface plasmon resonance (e.g., using a Biacore™ 3000 instrument and conditions as set out in Example 4.a. below) at 37° C.

In one embodiment of the invention, the equilibrium constant (KD) of the antibody with respect to binding of human IL-18 when measured by surface plasmon resonance (preferably using a Biacore™ T100 instrument and conditions as set out in Example 4.b. below) at 37° C. is less than 90 pM. In other embodiments of the invention, the equilibrium constant is 70 pM or less, 65 pM, 60 pM, 55 pM, or 50 pM, or less.

In a fourteenth aspect, this invention provides a humanised anti-interleukin-18 antibody, wherein said antibody displays a dissociation constant or off-rate (kd) of 0.0002 1/s or less with respect to binding of human IL-18 when measured by surface plasmon resonance (e.g., Biacore™, using a Biacore™ T100 instrument and conditions as set out in Example 4.b. below) at 37° C.

In a fifteenth aspect, this invention provides a humanised anti-interleukin-18 antibody, wherein said antibody comprises:

(a) a heavy chain comprising CDRs derived from a donor antibody, which CDRs have sequences set forth in SEQ ID NOs: 1, 2, and 3 grafted onto a heavy chain acceptor framework, wherein said heavy chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO: 37, wherein one or more residue/s of position/s 27, 28, 29, 93, 39, 40, 36, 71, 89, or 91 of the heavy chain is identical to the corresponding residue in the donor antibody heavy chain; and

(b) a light chain comprising CDRs derived from a donor antibody which CDRs have sequences set forth in SEQ ID NO: 4, 5, and 6 grafted onto a light chain acceptor framework which light chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO: 38, wherein position 71 and optionally one or more (e.g., all) residue/s of position/s 45, 83, 84, 85 of the light chain is identical to the corresponding residue in the donor antibody light chain.

In a sixteenth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

(a) a heavy chain comprising CDRs derived from a donor antibody which CDRs have sequences set forth in SEQ ID NOs: 1, 2, and 3 grafted onto a human heavy chain acceptor framework which heavy chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO: 37, wherein the residues at positions 27, 28, 29, 93 of the heavy chain are identical to the corresponding residues in the donor antibody heavy chain; and

(b) a light chain comprising CDRs derived from a donor antibody which CDRs have sequences set forth in SEQ ID NO:4, 5, and 6 grafted onto a light chain acceptor framework which light chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO:38, wherein residue at position 71 of the light chain of said anti-interleukin-18 antibody is identical to the corresponding residues in the donor antibody light chain.

In a seventeenth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

(a) a heavy chain comprising CDRs derived from a donor antibody which CDRs have sequences set forth in SEQ ID NOs: 1, 2, and 3 grafted onto a human heavy chain acceptor framework, wherein said heavy chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO: 37, wherein the residues at positions 27, 28, 29, 39, 40, and 93 of the heavy chain are identical to the corresponding residues in the donor antibody heavy chain; and

(b) a light chain comprising CDRs derived from a donor antibody which CDRs have sequences set forth in SEQ ID NOs: 4, 5, and 6 grafted onto a light chain acceptor framework, wherein said light chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO: 38, wherein the residue at position 71 of the light chain is identical to the corresponding residues in the donor antibody light chain.

In an eighteenth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

(b) a light chain comprising CDRs derived from a donor antibody which CDRs have sequences set forth in SEQ ID NOs: 4, 5, and 6 grafted onto a light chain acceptor framework, wherein said light chain acceptor framework comprises framework regions derived from the sequence set forth in SEQ ID NO: 38, wherein the residue at position 71 of the light chain is identical to the corresponding residues in the donor antibody light chain.

In a nineteenth aspect, this invention provides a humanised anti-interleukin-18 antibody comprising:

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